Stabilized preparations containing antibody

ABSTRACT

A stabilized antibody-containing preparation containing acetyltryptophan or an acetyltryptophan derivative or a salt thereof as a stabilizer for controlling the decrease in the biological activity of the antibody.

TECHNICAL FIELD

The present invention relates to antibody-containing preparations,particularly stabilized antibody-containing preparations in which thebiological activity of the antibody is not damaged even after long-termstorage.

BACKGROUND ART

With the development of genetic engineering technology, various proteinformulations became supplied in stable amounts. To ensure stability,these formulations are supplied in the dosage form of a lyophilizedprotein ingredient powder to be dissolved just before use in aseparately packaged water-soluble diluent or in the dosage form of aprotein solution formulation containing additives for improvingstability.

Recently, efforts have been made to provide various antibodypreparations in the form of solution formulations, but antibodies suchas immunoglobulins, monoclonal antibodies and humanized antibodies areunstable proteins liable to physical or chemical changes such asassociation or aggregation under stresses of filtration, concentrationand heating for removing viruses during the purification step. Antibodypreparations also have the disadvantage that the biological activity ofthe antibodies decreases after long-term storage though it is essentialfor them to maintain the binding activity for their antigens in vivo.

A stabilization effect was found by adding polymers including proteinssuch as human serum albumin or purified gelatin or oligomers such aspolyols, amino acids and surfactants as stabilizers for controllingchemical or physical changes. However, the addition of proteins such ashuman serum albumin or purified gelatin as stabilizers involves a verycomplicated step for removing viral contamination or other problems.

Acetyltryptophan was added to proteins such as albumin, human growthhormones and human B cell differentiation factor (BCDF) (JPB No.HEI7-68137, JPA No. HEI10-265404, JPA No. HEI3-27320). We previouslyreported stabilizing hematopoietic growth factors such as G-CSF and EPOby adding acetyltryptophan (PCT/JP01/01524).

However, it has not been so far known to stabilize antibody preparationsby adding acetyltryptophan. Moreover, all the conventional preparationsstabilized with acetyltryptophan described above were reported to showimproved protein residuals by adding acetyltryptophan, but its effect oncontrolling the decrease in the biological activity in proteinpreparations, especially antibody preparations has not been known.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide stable antibodypreparations in which high percentages of the antibody residual and thebiological activity of the antibody is highly maintained even afterlong-term storage.

As a result of careful studies to attain the above object, weaccomplished the present invention on the basis of the finding thatpreparations in which high percentages of the antibody residual and thebiological activity of the antibody is highly maintained even afterlong-term storage can be obtained by adding acetyltryptophan or anacetyltryptophan derivative or a salt thereof as a stabilizer.

Accordingly, the present invention provides a stabilizedantibody-containing preparation containing acetyltryptophan or anacetyltryptophan derivative or a salt thereof as a stabilizer forcontrolling the decrease in the biological activity of the antibody.

The present invention also provides a method for controlling thedecrease in the biological activity of the antibody in anantibody-containing preparation, comprising adding acetyltryptophan oran acetyltryptophan derivative or a salt thereof.

The present invention also provides a method for using acetyltryptophanor an acetyltryptophan derivative or a salt thereof for controlling thedecrease in the biological activity of the antibody in anantibody-containing preparation.

THE MOST PREFERRED EMBODIMENTS OF THE INVENTION

Antibodies used in the present invention are not specifically limited sofar as they bind to a desired antigen, and mouse antibodies, ratantibodies, rabbit antibodies, sheep antibodies, chimeric antibodies,humanized antibodies, human antibodies and the like can be used asappropriate. The antibodies may be polyclonal or monoclonal, butpreferably monoclonal because homogeneous antibodies can be stablyproduced. Polyclonal and monoclonal antibodies can be prepared byprocesses well known to those skilled in the art.

Hybridomas producing monoclonal antibodies can be basically constructedby known techniques as follows. A desired antigen or a cell expressing adesired antigen is used as an immunizing antigen to immunize host cellsaccording to a standard immunization technique, and the resultingimmunized cells are fused to known parent cells by a standard cellfusion technique, and then the fused cells are screened for monoclonalantibody-producing cells (hybridomas) by a standard screening method.Construction of hybridomas can be performed according to the method ofe.g. Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol.(1981) 73: 3-46). If the antigen has low immunogenicity, it can be boundto an immunogenic macromolecule such as albumin and used forimmunization.

Recombinant antibodies can be used, which are produced by transforming ahost with a suitable vector containing an antibody gene cloned from ahybridoma using genetic engineering techniques (for example, see Carl,A. K. Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES,Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).Specifically, the CDNA sequences for the variable regions (V regions) ofan antibody are synthesized from mRNA of a hybridoma using a reversetranscriptase. Thus obtained DNA sequences encoding the V regions of theantibody of interest are linked to the DNA sequences encoding theconstant regions (C regions) of the antibody of interest and integratedinto an expression vector. Alternatively, the DNA sequences encoding theV regions of the antibody can be integrated into an expression vectorcontaining the DNA sequences for the C regions of the antibody. They areintegrated into the expression vector in such a manner that they can beexpressed under the control of regulatory regions such as enhancers andpromoters. Then, a host cell can be transformed with this expressionvector to express the antibody.

In the present invention, recombinant antibodies, i.e. antibodiesartificially modified to reduce antigenicity in humans or to attainother purposes, such as chimeric antibodies and humanized antibodies canbe used. These modified antibodies can be prepared by known processes.Chimeric antibodies consist of the heavy and light chain variableregions of an antibody from a non-human mammal such as a mouse and theheavy and light chain constant regions of a human antibody and can beobtained by linking the DNA sequences encoding the variable regions ofthe mouse antibody to the DNA sequences for the constant regions of thehuman antibody and transforming a host with an expression vectorcontaining the linked sequences to allow it to produce a chimericantibody.

Humanized antibodies are also called reshaped human antibodies andobtained by grafting the complementarity-determining regions (CDRs) ofan antibody from a non-human mammal such as a mouse into thecomplementarity-determining regions of a human antibody and typical generecombination techniques for preparing them are also known.Specifically, DNA sequences designed to link the CDRs of a mouseantibody to the framework regions (FRs) of a human antibody aresynthesized by PCR from several oligonucleotides prepared to haveterminal overlapping regions. The resulting DNA sequences are linked tothe DNA sequences encoding the constant regions of the human antibodyand then integrated into an expression vector, which is transformed intoa host to allow it to produce a reshaped antibody (see European PatentPublication No. EP 239400, International Publication No. WO 96/02576).The FRs of the human antibody linked by the CDRs are selected in such amanner that the complementarity-determining regions form an appropriateantigen-binding site. If necessary, reshaped humanized antibodies mayhave some amino acid changes in the framework regions of the variableregions so that the complementarity-determining regions form anappropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993)53, 851-856).

Methods for obtaining human antibodies are also known. For example, adesired human antibody having a binding activity for a desired antigencan be obtained by in vitro immunizing human lymphocytes with thedesired antigen or a cell expressing the desired antigen and fusing theimmunized lymphocytes to human myeloma cells such as U266 (see JPB No.HEI1-59878). A desired human antibody can also be obtained by immunizinga transgenic animal having all human antibody gene repertoires with anantigen (see International Publications Nos. WO 93/12227, WO 92/03918,WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735). Methods forobtaining a human antibody by panning using a human antibody library arealso known. For example, phages binding to an antigen can be selected byexpressing the variable regions of a human antibody as single chainantibody fragments (scFv) on phage surfaces by a phage display method.The DNA sequences encoding the variable regions of the human antibodybinding to the antigen can be determined by analyzing the genes of theselected phages. A whole human antibody can be obtained by preparing asuitable expression vector on the basis of the determined DNA sequencesof the scFv fragments binding to the antigen. These methods have alreadybeen well known from WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236,WO 93/19172, WO 95/01438, WO 95/15388.

When an antibody is to be prepared by transforming a preliminarilyisolated antibody gene into a suitable host, the suitable host can beused in combination with an expression vector. Suitable eukaryotic cellsused as hosts include animal and plant cells and fungi. Known animalcells include (1) mammal cells such as CHO, COS, myeloma, BHK (babyhamster kidney), HeLa and Vero cells; (2) amphibian cells such asXenopus oocytes; or (3) insect cells such as sf9, sf21 and Tn5. Knownplant cells include cells of Nicotiana such as Nicotiana tabacum, whichcan be used as callus cultures. Known fungi include yeasts such asSaccharomyces spp., e.g. Saccharomyces serevisiae and filamentous fungisuch as Aspergillus spp., e.g. Aspergillus niger. Prokaryotic cells canbe used as producing systems using bacterial cells. Known bacterialcells include E. coli and Bacillus subtilis. Antibodies can be obtainedby transforming these cells with an antibody gene of interest andculturing the transformed cells in vitro.

Not only the host cells described above but also transgenic animals canbe used to produce recombinant antibodies. For example, an antibody geneis inserted into a gene encoding a protein produced specifically in milk(such as goat β casein) to prepare a fusion gene. A DNA fragmentcontaining the fusion gene bearing the antibody gene is injected intothe embryo of a goat and this embryo is implanted into a female goat. Anantibody is obtained from the milk produced by transgenic goats bornfrom the goat impregnated with the embryo or progeny thereof. Toincrease the amount of the antibody-containing milk produced by thetransgenic goats, hormones may be administered to the transgenic goatsas appropriate (see Ebert, K. M. et al., Bio/Technology (1994) 12,699-702).

Antibodies contained in stabilized preparations of the present inventioninclude, but not limited to, anti-tissue factor antibodies, anti-IL-6receptor antibodies, anti-HM1.24 antigen monoclonal antibodies,anti-parathyroid hormone related peptide antibodies (anti-PTHrPantibodies), etc.

Preferred reshaped humanized antibodies are humanized anti-tissue factorantibodies (see International Publication No. WO99-51743). Otherpreferred antibodies for use in the present invention include humanizedanti-IL-6 receptor antibodies (hPM-1) (see International Publication No.WO92-19759), humanized anti-HM1.24 antigen monoclonal antibodies (seeInternational Publication No. WO98-14580) and humanized anti-parathyroidhormone related peptide antibodies (anti-PTHrP antibodies) (seeInternational Publication No. WO98-13388).

Moreover, antibodies used in the present invention are not limited towhole molecule antibodies but may be antibody fragments or modifiedfragments and include divalent and monovalent antibodies so far as theybind to an antigen molecule to inhibit the activity of the antigen. Forexample, antibody fragments include Fab, (Fab′)₂Fv, Fab/c having one Faband a whole Fc, and single chain Fv (scFv) in which the heavy and lightchain Fv fragments are joined with a suitable linker.

Specifically, an antibody is treated with an enzyme such as papain orpepsin to produce antibody fragments or the genes encoding theseantibody fragments are constructed and introduced into an expressionvector and then expressed in a suitable host cell (for example, see Co,M. S. et al., J. Imunol. (1994) 152, 2968-2976, Better, M. & Horwitz, A.H. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc.,Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496,Academic Press, Inc., Lamoyi, E, Methods in Enzymology (1989) 121,652-663, Rousseaux, J. et al, Methods in Enzymology (1989) 121, 663-669,Bird, R. E. et al., TIBTECH (1991) 9, 132-137).

Fragments scFv are obtained by joining the heavy chain variable regionand the light chain variable region of an antibody. In the scFvfragments, the heavy chain variable region and the light chain variableregion are joined by a linker, preferably a peptide linker (Huston, J.S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). Theheavy chain variable region and the light chain variable region in thescFv fragments may be derived from any antibody described herein. Thepeptide linker used for joining the variable regions is e.g. asingle-stranded peptide consisting of 12-19 amino acid residues.

The DNA sequences encoding the scFv fragments are obtained by PCRamplification using the entire sequences of the DNA encoding the heavychain or the heavy chain variable region and the DNA encoding the lightchain or the light chain variable region of the antibody or a partthereof encoding a desired amino acid sequence as templates incombination with primer pairs defining both ends of these sequences andthen using the DNA encoding a peptide linker region in combination witha primer pair defining both ends thereof to be linked to the heavy andlight chains.

Once the DNA sequences encoding the scFv fragments are prepared, anexpression vector containing these fragments and a host transformed withthe expression vector can be obtained by conventional methods, and thehost can be used to give scFv by conventional methods.

These antibody fragments can be produced by the host after obtaining andexpressing the genes for them in the same manner as described above. Asused herein, the “antibody” also means to include these antibodyfragments.

Modified antibodies including antibodies conjugated with variousmolecules such as polyethylene glycol (PEG) can also be used. As usedherein, the “antibody” also means to include these modified antibodies.Such modified antibodies can be obtained by chemically modifying theantibodies obtained as above. Methods for modifying antibodies havealready been established in this field of art.

Antibodies used in the present invention may also be bispecificantibodies. Bispecific antibodies may have antigen-binding sitesrecognizing different epitopes of an antigen molecule or may have oneantigen-binding site recognizing an antigen molecule and anotherantigen-binding site recognizing a cytotoxin such as a chemotherapeuticagent or a cell-derived toxin. In this case, the cytotoxin can directlyact on cells expressing the antigen molecule to specifically damagecancer cells so that the growth of the cancer cells can be inhibited.Bispecific antibodies can be prepared by joining HL pairs of twoantibodies, or fusing hybridomas producing different monoclonalantibodies to prepare a fusion cell producing a bispecific antibody.Bispecific antibodies can also be prepared by genetic engineeringtechniques.

Antibodies contained in stabilized preparations of the present inventionmay belong to any immunoglobulin class, preferably IgG such as IgG1,IgG2, IgG3 and IgG4, more preferably IgG4.

As used herein, the antibody-containing sample may be a samplecontaining any antibody whether natural or recombinant, preferably aculture medium obtained after culturing antibody-containing mammaliancells such as CHO cells or further applying some treatment such aspartial purification.

As used herein, the “biological activity of the antibody” means thebinding activity of the antibody for its antigen or various biologicalactivities generated by binding of the antibody to an antigen moleculesuch as neutralization activity, antagonist activity and agonistactivity against the antigen molecule. The expression “controlling thedecrease in the biological activity of the antibody” means maintaining80% or more, preferably 90% or more of the biological activity of theantibody in the antibody formulation just prepared. The biologicalactivity of the antibody can be determined by assays such as ELISA orusing a surface plasmon sensor or the like.

Preferably, antibody preparations of the present invention aresubstantially free from proteins such as human serum albumin or purifiedgelatin as stabilizers.

Acetyltryptophan or acetyltryptophan derivatives or salts thereof usedas stabilizers in the present invention include free acetyltryptophan oracetyltryptophan derivatives and salts thereof such as sodium salts,potassium salts and hydrochlorides. Acetyltryptophan or acetyltryptophanderivatives or salts thereof used in the preparations of the presentinvention may be D-, L- or DL-isomers, more preferably L-isomers.Acetyltryptophan derivatives include, but not limited to,acetyltryptophan methyl ester, acetyltryptophan ethyl ester,acetyltryptophan propyl ester, acetyltryptophan amide,chloroacetyltryptophan, etc.

The amount of acetyltryptophan or acetyltryptophan derivatives or saltsthereof added to the preparations of the present invention depends onthe nature and concentration of the antibodies used, dosage form(freeze-dried formulations or solution formulations) and the derivativesused. They are typically contained in freeze-dried formulations at afinal dose of 0.1-300 mM, preferably 1-200 mM, more preferably 1-100 mM.They are typically contained in solution formulations at a final dose of0.1-30 mM, preferably 0.5-20 mM, more preferably 0.5-10 mM. In the caseof freeze-dried formulations, stable antibody preparations withexcellent reconstitution can be provided at 0.1-30 mM, preferably 1-30mM, more preferably 1-10 mM.

The weight ratio of antibodies to acetyltryptophan is generally100:1-0.1:1. In the case of humanized anti-tissue factor antibodies, itis preferably 10:1-0.5:1, more preferably 2:1-1:1.

When the antibody is e.g. a humanized anti-tissue factor antibody,antibody preparations of the present invention show a residual ofhumanized anti-tissue factor antibody of 90% or more, preferably 95% ormore and a residual of biological activity of 80% or more, preferably90% or more after accelerated testing at 40° C. for 1 month.

Antibody preparations preferably have a pH of 4-8, more preferably5-7.5. However, the pH depends on the antibody contained and is notlimited to these values. In the case of e.g. humanized anti-tissuefactor antibodies, the pH is preferably 4-7, more preferably 5-6. The pHcan be adjusted with NaOH or the like, or basic amino acids such ashistidine, arginine and lysine or basic amino acid derivatives or saltsthereof to reduce antibody aggregation.

Preparations of the present invention may contain isotonizing agents,e.g., polyethylene glycol; and sugars such as dextran, mannitol,sorbitol, inositol, glucose, fructose, lactose, xylose, mannose,maltose, sucrose and raffinose.

Preparations of the present invention may further contain surfactants.Typical examples of surfactants include:

nonionic surfactants, e.g., sorbitan fatty acid esters such as sorbitanmonocaprylate, sorbitan monolaurate, sorbitan monopalmitate; glycerinfatty acid esters such as glycerin monocaprylate, glycerinmonomyristate, glycerin monostearate; polyglycerin fatty acid esterssuch as decaglyceryl monostearate, decaglyceryl distearate, decaglycerylmonolinoleate; polyoxyethylene sorbitan fatty acid esters such aspolyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitanmonooleate, polyoxyethylene sorbitan monostearate, polyoxyethylenesorbitan monopalmitate, polyoxyethylene sorbitan trioleate,polyoxyethylene sorbitan tristearate; polyoxyethylene sorbitol fattyacid esters such as polyoxyethylene sorbitol tetrastearate,polyoxyethylene sorbitol tetraoleate; polyoxyethylene glycerin fattyacid esters such as polyoxyethylene glyceryl monostearate; polyethyleneglycol fatty acid esters such as polyethylene glycol distearate;polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether;polyoxyethylene polyoxypropylene alkyl ethers such as polyoxyethylenepolyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propylether, polyoxyethylene polyoxypropylene cetyl ether; polyoxyethylenealkyl phenyl ethers such as polyoxyethylene nonyl phenyl ether;polyoxyethylene hardened castor oils such as polyoxyethylene castor oil,polyoxyethylene hardened castor oil (polyoxyethylene hydrogenated castoroil); polyoxyethylene beeswax derivatives such as polyoxyethylenesorbitol beeswax; polyoxyethylene lanolin derivatives such aspolyoxyethylene lanolin; polyoxyethylene fatty acid amides such aspolyoxyethylene stearic acid amide having an HLB of 6-18;

anionic surfactants, e.g., alkyl sulfates having a C10-18 alkyl groupsuch as sodium cetyl sulfate, sodium lauryl sulfate, sodium oleylsulfate; polyoxyethylene alkyl ether sulfates having an average EO molenumber of 2-4 and a C10-18 alkyl group such as sodium polyoxyethylenelauryl sulfate; alkyl sulfosuccinic acid ester salts having a C8-18alkyl group such as sodium laurylsulfosuccinate; and

natural surfactants, e.g., lecithin; glycerophospholipids;sphingophospholipids such as sphingomyelin; sucrose fatty acid esters ofC12-18 fatty acids. Preparations of the present invention can containone or more of these surfactants in combination.

Preferred surfactants are polyoxyethylene sorbitan fatty acid esters,more preferably Polysorbates 20, 21, 40, 60, 65, 80, 81, 85, mostpreferably Polysorbates 20 and 80.

The amount of surfactants to be added to antibody preparations of thepresent invention is typically 0.0001-10% (w/v), preferably 0.001-5%,more preferably 0.005-3%.

Antibody preparations of the present invention may further containdiluents, solubilizing agents, excipients, pH-modifiers, soothingagents, buffers, sulfur-containing reducing agents, antioxidants or thelike, if desired. For example, sulfur-containing reducing agents includeN-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol,thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid andsalts thereof, sodium thiosulfate, glutathione, andsulfhydryl-containing compounds such as thioalkanoic acid having 1 to 7carbon atoms. Antioxidants include erythorbic acid,dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherolacetate, L-ascorbic acid and salts thereof, L-ascorbyl palmitate,L-ascorbyl stearate, sodium bisulfite, sodium sulfite, triamyl gallate,propyl gallate or chelating agents such as disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate and sodium metaphosphate.Other components commonly added may also be contained, e.g., inorganicsalts such as sodium chloride, potassium chloride, calcium chloride,sodium phosphate, potassium phosphate and sodium bicarbonate; andorganic salts such as sodium citrate, potassium citrate and sodiumacetate.

Preparations of the present invention can be prepared by dissolvingthese components in an aqueous buffer known in the field of solutionformulations such as a phosphate buffer (preferably sodium monohydrogenphosphate—sodium dihydrogen phosphate system) and/or a citrate buffer(preferably sodium citrate buffer) to prepare a solution formulation, orfreeze-drying or spray-drying thus prepared solution formulation bystandard procedures. In the case of solution preparations, antibodiesare preferably dissolved in a glycine buffer and/or histidine buffer toinhibit heat-induced aggregation. Aggregation can be further inhibitedby adding glycine and/or sucrose.

Stabilized antibody preparations of the present invention are normallyadministered via parenteral routes such as injection (e.g. subcutaneous,intravenous or intramuscular injection) or percutaneous, mucosal, nasalor pulmonary administration, but may also be orally administered.

Stabilized preparations of the present invention may be in the form ofsolution formulations or freeze-dried formulations to bedissolved/reconstituted before use. Suitable excipients forfreeze-drying include sugar alcohols or sugars such as mannitol orglucose.

The amount of antibodies contained in preparations of the presentinvention depends on the type of the disease to be treated, the severityof the disease, the age of the patient and other factors, but generallycorresponds to a final concentration of 0.1-200 mg/ml, preferably 1-120mg/ml.

The following examples further illustrate the present invention without,however, limiting the scope of the invention thereto. Various changesand modifications can be made by those skilled in the art on the basisof the description of the invention, and such changes and modificationsare also included in the present invention.

EXAMPLES Example 1 Effects of Acetyltryptophan on Residual of HumanizedAnti-Tissue Factor Antibody and Biological Activity

(1) Materials

The humanized anti-tissue factor antibody is the humanized antibodyprepared in Example 4 of International Publication No. WO99-51743 andbelongs to immunoglobulin class IgG4.

(2) Test Samples

Formulated solutions containing 2 mg/mL humanized anti-tissue factorantibody, 20 mmol/L sodium citrate buffer (pH 5), 10 mmol/L sodiumchloride and 0, 1.25 or 2.5 mg/mL acetyltryptophan were prepared andaseptically filtered, and then a vial was aseptically packed withprecisely 1 mL of each solution. The humanized anti-tissue factorantibody used was a recombinant humanized anti-tissue factor antibodyobtained with CHO cells, and the acetyltryptophan added was an L-isomer.

Thus aseptically prepared solution formulations containing the humanizedanti-tissue factor antibody were used as test samples after acceleratedtesting in an incubator at 40° C. for 1 month.

(3) Method for Determining the Antibody Content and Calculating theAntibody Residual.

The humanized anti-tissue factor antibody content in each sample wasdetermined by gel permeation chromatography (GPC) using 10 mmol/L sodiumphosphate buffer, pH6.8/150 mmol/L sodium chloride as a mobile phase ata flow rate of 0.7 mL/min on a column (TOSOH G3000SW_(XL)).

The humanized anti-tissue factor antibody content determined by thismethod was used to calculate the antibody residual (%) after acceleratedtesting according to the following equation.

${{Antibody}\mspace{14mu}{residual}\mspace{14mu}(\%)} = {\frac{\begin{matrix}\left( {{Humanized}\mspace{14mu}{anti}\text{-}{tissue}\mspace{14mu}{factor}\mspace{14mu}{antibody}} \right. \\\left. {{content}\mspace{14mu}{after}\mspace{14mu}{acceleration}\mspace{14mu}{for}\mspace{14mu} a\mspace{14mu}{given}\mspace{14mu}{period}} \right)\end{matrix}}{\begin{matrix}\left( {{Humanized}\mspace{14mu}{anti}\text{-}{tissue}\mspace{14mu}{factor}\mspace{14mu}{antibody}} \right. \\\left. {{content}\mspace{14mu}{in}\mspace{14mu}{unaccelerated}\mspace{14mu}{formulation}} \right)\end{matrix}} \times 100}$

(4) Assay Protocol for Measuring the Biological Activity of the Antibody

The biological activity of the humanized anti-tissue factor antibody wasassayed as follows. The antigen against which the humanized anti-tissuefactor antibody is directed is tissue factor which promotes coagulationin tissue fluid and which is also called blood coagulation factor III ortissue thromboplastin. Tissue factor is a cofactor for blood coagulationfactor VII initiating extrinsic coagulation and forms a molecularcomplex with factor VII or activated factor VIIa to activate factor Xand factor IX. Thus, the biological activity of the humanizedanti-tissue factor antibody can be determined by reacting antibodysamples with blood coagulation factors III, VIIa and X to produce factorXa and cleaving the factor Xa by its substrate to measure the absorbanceof the color developed.

Reagents

Thromborel S: supplied by Dade Behring (a reagent containing coagulationfactor III).

Factor VIIa: supplied by CALBIOCHEM.

Factor X: supplied by Enzyme Research.

Chromogenic substrate Testzyme S-2222: supplied by Daiichi PureChemicals.

TBS: supplied by TAKARA.

CaCl₂: commercial grade.

BSA: supplied by Sigma.

Hexadimethrine bromide: supplied by Sigma.

EDTA: commercial grade.

Preparation of the Reagents

Thromborel S: Thromborel S (200 mg/vial) is dissolved in 4 mL/vial ofMilli-Q water. The solution is dispensed into tubes at 100 μL/tube andthen stored at −80° C. Before use, the frozen solution is thawed at roomtemperature and then heated at 37° C. for 15 minutes.

Factor VIIa (coagulation factor): prepared at 500 PEU/mL in Assay Buffer(hereinafter referred to as A.B.). The solution is dispensed into tubesat 20 μL/tube and then stored at −80° C.

Factor X (coagulation factor): prepared at 25 PEU/mL in A.B. Thesolution is dispensed into tubes at 100 μL/tube and then stored at −80°C.

Chromogenic substrate S-2222: dissolved in 17 mL of Milli-Q water andstored at 4° C.

EDTA: prepared at 500 mM in Milli-Q water.

Polybrene solution: Hexadimethrine bromide is dissolved at 0.6 mg/mL inMilli-Q water.

Preparation of the Buffer and Solutions

Assay Buffer (A.B.): TBS (pH 7.6) containing 5 mM CaCl₂, 0.1% BSA.

Mixed solution of Factor VIIa & Thromborel S: a solution of Factor VIIaand Thromborel S diluted in A.B. to final concentrations of 0.1 PEU/mLand 1:120 (v/v), respectively.

Factor X solution: a solution of Factor X diluted in A.B. to a finalconcentration of 0.25 PEU/mL.

100×standard solution: a solution of the humanized anti-tissue factorantibody prepared at 180 μg/mL in A.B.

100×sample solution: a solution of the humanized anti-tissue factorantibody in each sample prepared at 120 μg/mL in A.B.

Mixed chromogenic substrate S-2222 solution: prepared at a ratio ofchromogenic substrate S-2222: polybrene solution=1:1.

Method

1. Dispense the mixed solution of factor VIIa & Thromborel S into aplate at 60 μL/well and allow the plate to stand at room temperature for60 minutes.

2. Dilute each of 100×standard solution and 100×sample solution 100-foldin Factor X solution.

3. Further dilute the standard solution to 1800 ng/mL -356 ng/mL and thesample solution to 1200 ng/mL-533 ng/mL in Factor X solution at aconcentration ratio between the diluted solutions of 1.5.

4. Dispense the diluted sample into the plate at 40 μL/well and allowthe plate to stand for 30 minutes.

5. Stop the reaction by adding 10 μL/well of 500 mmol/L EDTA solution,and then dispense the mixed chromogenic substrate S-2222 solution intothe plate at 50 μL/well and allow the plate to stand at room temperaturefor 60 minutes.

6. Measure the absorbance at 405 nm-655 nm.

(5) Results

The results are shown in Table 1.

TABLE 1 Antibody residual Concentration of after 1 month @ 40° C.acetyltryptophan [% of Initial] [mg/mL] GPC Biological activity 0 97.982.0 1.25 (5 mM) 99.1 99.0

As shown from Table 1, the sample containing acetyltryptophan showed aremarkable stabilization effect with little decrease in biologicalactivity while maintaining a high residual of antibody.

1. A stabilized antibody-containing preparation containing an antibody and acetyltryptophan or an acetyltryptophan derivative or a salt thereof as a stabilizer capable of controlling the decrease in the biological activity of the antibody, wherein the antibody belongs to immunoglobulin class IgG and the preparation is substantially free from proteins as stabilizers.
 2. The stabilized antibody-containing preparation of claim 1 wherein the stabilizer is acetyltryptophan or a salt thereof.
 3. The stabilized antibody-containing preparation of claim 1 wherein the antibody is a chimeric antibody, humanized antibody or human antibody.
 4. The stabilized antibody-containing preparation of claim 1 wherein the antibody is an antibody fragment, modified antibody or modified antibody fragment.
 5. The stabilized antibody-containing preparation of claim 1 wherein the antibody is an anti-tissue factor antibody.
 6. The stabilized antibody-containing preparation of claim 1 wherein the antibody is an anti-tissue factor antibody and the anti-tissue factor antibody is a humanized anti-tissue factor antibody.
 7. The stabilized antibody-containing preparation of claim 1 in the form of a freeze-dried formulation.
 8. The stabilized antibody-containing preparation of claim 1 in the form of a solution formulation.
 9. The stabilized antibody-containing preparation of claim 1 wherein acetyltryptophan or an acetyltryptophan derivative or a salt thereof is contained at 1-100 mM. 